棉花GhMYB44, GhAP2和GhARC基因功能鉴定及CRISPR/Cas9全基因组脱靶分析

发布时间：2021-12-17 19:50

　　棉花是世界上最重要的经济作物之一,棉花纤维是全球经济的基础和纺织业的支柱,其他副产品可以作为牲畜饲料、棉籽油、肥料、纸张及其他消费品,另外,棉花可以作为一种理想的植物,对基因组进化、多倍体化和单细胞生物学等进行不同的生物学研究。MYB转录因子包含一个MYB域,由52个氨基酸基序的4个不完全重复序列组成,该转录因子在保卫细胞信号转导中起着重要作用。AtMYB44是一种多面转录因子,主要与拟南芥乙烯信号通路相关。APETALA2/乙烯反应因子（AP2/ERF）是植物特异性转录因子（TFs）的一个超家族,它在调节植物器官发育,不同环境和生物胁迫的反应中起着至关重要的作用。NB-ARC结构域是一种调节程序性细胞死亡的STAND（信号转导多个结构域的ATP酶）蛋白,能够与ATP结合进行水解并诱导下游阻力信号传导。基因组编辑（GE）是通过编辑不同生物的基因组序列从而改变生物特性的一种方法。近年来,从简单到复杂的基因组,人们已经探索了各种各样的基因编辑工具,CRISPR（成簇的有规律的间隔的短回文重复序列）是最新并且功能最为强大的一种编辑技术,它的应用彻底改变了科学界。CRISPR/CAS9是一种R... 

【文章来源】：华中农业大学湖北省 211工程院校 教育部直属院校

【文章页数】：157 页

【学位级别】：博士

【文章目录】：
摘要
Abstract
List of abbreviations
CHAPTER 1
    1 Introduction
        1.1 Cotton production and insect attack
            1.1.1 The interaction and defense of plants against insect stress
        1.2 Transcription factors(TFs)
            1.2.1 MYB transcription factors
                1.2.1.1 MYB44 gene
            1.2.2 APETALA2/Ethylene-Responsive Factor(AP2/ERF)gene
            1.2.3 Nucleotide-binding(NB)-ARC domain-containing disease resistanceprotein
        1.3 Whole genome sequencing to detect on and off-targets
        1.4 Genome editing techniques and their applications
            1.4.1 Genome editing
                1.4.1.1 The CRISPR/Cas system
                    1.4.1.1.1 The mechanism of the CRISPR/Cas9 system
                    1.4.1.1.2 Methodology for the screening of CRISPR/Cas9-induced mutant
                    1.4.1.1.3 The diverse applications of CRISPR/Cas9 system in major crops,Vegetables and fruits
        1.5 Aims of the study
CHAPTER 2
    2 Materials and methods
        2.1 Sequence analysis of GhMYB44 and GhAP2 and GhARC genes
        2.2 Subcellular localization of the GhMYB44 gene
        2.3 Computational sgRNA design and selection
        2.4 Vector construction for CRISPR/Cas9 and overexpression
        2.5 Plant material for Agrobacterium-mediated transformation in cotton
        2.6 Initiation,maintenance of embryogenic calli and plant regeneration
        2.7 Plant material,growth conditions and measurement of different agronomictraits
        2.8 DNA extraction and Southern blotting
        2.9 RNA extraction,RT-PCR,and qRT-PCR
        2.10 Tissue culturing and generation of homozygous transgenic lines
        2.11 On-target analysis of CRISPR/Cas9 edited plants
        2.12 Genomic DNA isolation and library construction for WholeGenome Sequencing
        2.13 Bioinformatics analysis for variant calling
        2.14 Genome-wide prediction off-target cleavage sites and detectiontheir mutations
        2.15 Analysis genetic variation of potential off-targets and PAMs between WTand cotton reference genome
        2.16 Data availability
CHAPTER 3
    3 RESULTS
        3.1 Characteristics of GhMYB44,GhAP2,and Gh ARC genes
        3.2 Knockout of GhMYB44,GhAP2,and GhARC genes and overexpression ofGhMYB44 gene in cotton
        3.3 Confirmation of GhMYB44,GhAP2,and GhARC transgenes and generationof homozygous lines
        3.4 Subcellular localization of GhMYB44 gene and Expression analysis ofGhMYB44,GhAP2,and GhARC genes
        3.5 Measurement of agronomic traits of GhMYB44,GhAP2,and GhARC genes
        3.6 Whole genome sequencing of CRISPR/Cas9-edited cotton plants,wild-typeand negative plants
        3.7 Detection of on-target mutations at the genome-wide scale
        3.8 Inherent genetic variation or/and somaclonal variation in Cas9-edited plants,wild-type,and negative plants
        3.9 Low frequency off-target mutations detected in Cas9-edited plants
        3.10 Genetic variation among cultivars can generate novel off-target sites
        3.11 Investigation of spontaneous mutations and the inheritance of Cas9-editedmutations
CHAPTER 4
    4 Discussion
Conclusion and future perspectives
REFERENCES
Appendices
Acknowledgements



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